The present invention relates to a new hydrophobe biomolecular structure, a method for the preparation of the structure and use of the new structure for the manufacture of a medicament.
When a cell should be intro-transfected with DNA and/or a plasmid the molecule should not be bigger than 10 nm and have hydrophobic properties. It also must be stable enough so that the plasmid/DNA can be handled.
The administration of a peptide to a patient is normally performed by injection, as biological uptake of a peptide by other routes of administration is difficult. The molecule to be introduced should not larger than 10 nm and have hydrophobic properties.
Hyaluronic acid (Hy) is a naturally occurring glycosaminoglycan consisting of a linear polymer of repeating units of glucuronic acid and N-acetyl-glucosamine. The molecular weight can vary over a wide range depending on the source. Hy is present in several tissues of animals, and in some organs, such as rooster combs, in concentrations high enough for commercial scale extraction. Such tissue contains Hy of a wide range of molecular weights and during a complex series of extraction, purification and sterilisation steps, high molecular weight chains are more or less degraded resulting in a final product having a considerably narrower molecular weight range.
Hy is non-toxic and is decomposed in the body and thus suitable for pharmacological use.
A commercial available hyaluronic acid product is HEALON(copyright) (Kabi Pharmacia AB, Uppsala, Sweden) which has a average molecular weight of about 4000000 Dalton. This product is produced as outlined in U.S. Pat. No. 4,141,973 and is an ultra-pure product. There are many literature references relating to the use of viscoelastic products of HA in ophthalmologic application and the preparation of such products, including the preparation of chemically modified HA.
Hy is known in slow release formulations and in WO 9005522 HA is mentioned as a slow release carrier together with a binding protein for e.g. CH or IGF.
Low molecular weight hyaluronic acid LMWHA is known as carrier for pharmaceutical active agents and for pharmaceutical activity by itself.
EP 522 491 discloses a freeze-dried composition comprising hyaluronic acid arid a polypeptide, which is administered by injection after reconstitution of the composition.
A patent application, WO 97/15330, claiming Hyaluronic acid as DNA carrier for gene Therapy and VEGF antisense DNA to treat abnormal retinal vascularisation was published May 1, 1997.
Hyaluronic acid molecular mass 300-5000 kDalton is claimed to increase viral vectors uptake by adjuvant effect page 33.
The mechanisms is suggested to be of targeting nature, that is, Hy binds to the cell receptor and to an adeno-virus construct increasing the contact time which facilitate and increase the efficiency of transfection page 55.
The goal of the compaction of biomolcules is to improve the oral bioavailability of peptides and thereby avoid the need of parental administration.
The used procedure will also improve the transfection efficiency of genes into mammalian cells for a stable expression and thereby avoid the need of toxic plasmid-DNA complex resulting in a short living expression and in side effects.
Full biological effect of compact peptide is demonstrated in biological assay and in vivo. The size and the durable hydrophobic properties obtained at pH 6 of the peptide suggest an improved oral bioavailability. Plasmid-DNA complex has been compact from 87 nm to 7.5 nm suggesting an improved transfection efficiency of genes.
Peptides are compacted from 2.4 nm to  less than 0.3 nm, improving its ability to penetrate across biological membranes e.g. the gut wall and the stratum corneum. The plasmid-DNA construct is compacted from 87 nm to  less than 10 nm, improving the DNA-structure for free passage through the nuclear pore. These new hydrophobic structures are obtained by pH-shifts, by elimination of molecular charge and bindings, by evacuation of water and ions, and finally stabilised with Hyaluronan.
These compacted structures can be administrated by new routes and thereby avoid the need of parental administration. Examples of these structures are peptides such as growth factors, metabolic regulators such as insulin and the like, plasmid-DNA and related bio-active molecules such as naked DNA, RNA and poly-electrolyte structures such us heparin and heparin derivatives. The routes include oral-, pulmonary-, nasal-, topical administration and intra cellular trafficking. These routes will make drug application more convenient especially for children. The drug will also be easier to handle which will result in an improved compliance. The cost of production for most of these drugs will be less since there are no requirements for sterile production of drugs administered the suggested way.
Pharmaceutical research is today devoted to improve the ability of the drug to penetrate biological membrane. One approach is to entrap bio-molecules in carrier system such as bioadhesive gels, liposomes or micro-beads.
Another approach is to replace peptides and related biological molecules with organic compounds designed for oral delivery and with a molar mass less than 500 Dalton. These structures are screened for activation of soluble receptors in tracks with around 96 probes. In about 75000 structures can easily be evaluated in a short time. Up til now however no active structure has been found although the screening started for around 10 years ago.
Compacting of well-known peptides such as growth hormones and insulin as well as uncomplicated DNA structures will eliminate expansive and extended toxicological studies. Clinical studies of these compacted structures will also be less complicated than for those studies required for new chemical entities and for entrapped bio-structures in liposome, micro-spheres and related carrier systems.
Thus, one embodiment of the present invention is a hydrophobe biomolecular structure containing a polymer and a polar biostructure. The structure is derived from collapsing the structure, when the structure is made to pass over the structure""s point of collapse. The result will be a compacted hydrophobic structure with buried polar groups and a minimum size, and the polymer is surrounding the biostructure.
In a prefered embodyment of the inventive hydrophobic biomolecular structure is:
the polymer a glycosaminoglycan such as hyaluronic acid or a cationic polysaccharide such as chitosan; and
the polar biostructure a nucleotide such as plasmid-DNA, DNA or RNA, a peptide such as insulin, growth hormone, recombinat growth hormone, heparin, heparin derivatives or enzyme, or a monoclonal.
Another embodiment of the present invention is a method for the production of the hydrophobic biomolecular structure. The method includes the steps of:
a) solubilisation of the polymer and solubilisation of the polar biostructure by addition of an acid;
b) collapsing the polar biostructure by addition of an electrolyte by passing the polar biostructure over polar biostructure""s point of collapse and changing the hydrodynamic randii to a minimum compact size; and
c) dialysing the biomolecular structure, thereby obtaining a compacted hydrophobic structure with the polar groups buried and the polymer surrounding the biostructure.
In a prefered embodiment of the inventive method the acid in step a) is hydrochloric acid, sulphuric acid, phosphoric acid or acetic acid and the pH is less than 3, preferable in the range of 1.0 to 2.5.
In another prefered embodiment of the inventive method the electrolyte of step b) contains cations such as NH4+, K+, Na+, Ca2+, Mg2+, Zn2+, Fe2+, Fe3+and anions such as sulfates, chloride, acetates.
In another embodiment of the present invention the hydrophobic biomolecular structure is used as a medicament.
Preferred embodiments and other aspects of the present invention are defined in the independent and the dependent claims.
Hy is normally in the form of loops, but to our surprise, we have found that it can become straight in the presence of protons, H+.
Thus, when HCl is added to a solution of Hy, the molecules will become straight and will be positively charged.
By Hy is here meant Hyaluronan with a molecular weight of 150 kDa in a range of 80-360 kDa.
An optimal compaction (=size) and hydrophobic properties of plasmid ( less than 10 nm) and peptides ( less than 0.3 nm) are found by elimination of charge and molecular bindings by addition of HCl to pH less than 2 and of different ions. These ions are cationic ions: NH4+, K+, Na+ and anionic ions: sulfates, chloride, carbonates, acetates.
A stable complex/polymer is formed by Hy surrounding the plasmid, the peptides respectively when Hy is changing its structure back to a curling like structure when pH is changed to pH 6. It is then possible to dilute the solutions to the desired strength.
I. Plasmids
Historically transfection of plasmid/DNA into cells has been performed by either using a signal substance or by passive diffusion. A compaction=compression of plasmid, which are about 60 to 500 nm, results in a much smaller molecules. Transfection with small molecules has earlier been performed with the use of lipid-amine-complexes or sodium chloride. However, these amines result in a toxic complex for the cell and are thus not suitable. When NaCl alone is used, the complex is not stable.
The compaction according to our invention occurs when NaCl or Na2SO4 and HCl are mixed with the plasmid. The water, which was included in the plasmid, is evacuated and hydrogen bridges are eliminated.
The molecules become more negatively charged and more hydrophobic.
When the straightened Hy and the compressed plasmid are mixed at low pH and dialysed to a pH of about 6.5 a stable complex is formed with a diameter of about 7.5 nm.
This complex is stable for at least 3 month in aqueous solution and is non-toxic.
II Peptides
Recombinant growth hormone, rhGH is here used as an example of a peptide of great medical interest.
rhGH has a size of about 2.4 nm and cannot be given orally because of its size, its hydrophobic properties and its ease to be bio-graded. This unable rhGH like most other peptides to pass bio-membranes. When rhGH is in a solution at its isoelectric point the molecule is not charged and is hydrophobic. If straightened Hy is added, complexes are formed in which the rhGH molecule is xe2x80x9csurroundedxe2x80x9d by Hy and the particles are less than 1 nm. These particles appear stable and could be administered orally.
Method of Compaction
By changing pH to a strong acid solution (pH 1.5) peptides gets easily soluble and Hy becomes a charged structure stretching out from a curling cylinder to straight line. By adding electrolytes to the solution a minimum of charge occurs in the peptide. This results in a total collapse of the peptide structure as it passes its pI (isoelectric point).In regulating the ions (NaCl and Na2SO4) and the rhGH concentrations with the speed of the pH-change when passing the pI. The hydrodynamic radii of the particles in the dispersion of the peptide are changed from 2.4 to 0.22 nm. Hy stabilise the dispersion in changing its structure back to a curling like structure when pH is changed to pH 6. It is then possible to dilute the dispersions to the concentration desired. The hydrophobic properties of the peptide structure is found to be optimal as pH is changed in a strong acid solution pH less than 2 and by dialyses transferred to a neutral solution pH 6.
HI-HPLC assays of none compact (n-compact.) and compact rhGH suggest activity of compact rhGH to be within acceptable limits and of the same magnitude as for of none compact rhGH. No agglomerates or particles are found upon dilution. Parental administration in H-x rats of Compact rhGH resulted in a dose response of growth gain and of Tibia elongation. None compact rhGH also resulted in a dose response of growth gain and of Tibia elongation in H-x rats. The growth gain and the Tibia elongation were of similar magnitude for compact and none compact rhGH.
No growth gain or Tibia elongation was recorded for Placebo.
Conclusion
The structure of rhGH has been compacted. One rhGh unit has been compact from radii of 2.4 nm to 0.22 nm.
Full biological effect of compact rhGH is demonstrated in vivo. Hydrophobic properties of rhGH is obtained in acid solutions pH less than 2 and a durable hydrophobic structure is demonstrated when pH is changed to pH 6. The size and the hydrophobic properties of the complex suggest an improved oral bioavailability of rhGH.